The protozoan, Giardia lamblia, belongs to the archezoan subkingdom. Some primitive organisms of this subkingdom including Giardia have been found to utilize pyrophosphate (PPi) rather than ATP as an energy source in certain glycolytic reactions. It is hypothesized that these glycolytic enzymes represent 'metabolic fossils' and that PPi may have served as primitive high energy compound. The overall objective of the proposed work is to characterize these PPi dependent enzymes so as to understand the specific molecular structures that result in the preferential utilization of the high energy phosphoryl bond of PPi vs that of ATP. The proposed work will be focused on detail characterization of two such enzymes that have been detected in Giardia, a PPi dependent phosphofructokinase and a pyruvate phosphate dikinase. The specific aims include isolation and characterization of the molecular weight properties, kinetic parameters and regulation by metabolic effectors on these two enzymes. These analyses could further our understanding of energy transfer through phosphoryl bond hydrolysis using the simplest high energy phosphate compound like PPi. Structural characterization of the two enzymes will involve probing the PPi binding and catalytic domains using potential affinity analogs of PPi like azidonitrophenyl pyrophosphate, ferrate ion and pyridoxal phosphate. Chemical modification and steady state kinetics will be used to analyze the binding and catalysis of PPi and ATP. These affinity labels will be used as probes to isolate tryptic peptides at or around the PPi binding and catalytic sites by reverse phase HPLC. The amino acid sequences of these peptides will be determined by automated gas phase sequence analysis. The purpose will be to understand the nature of PPi binding and catalysis in terms of enzyme structure and function. The second objective is to determine if there are similarities in the PPi binding site and the ATP binding sites of higher eukaryotes which will provide evidence for the hypothesis that enzymes utilized PPi as a primitive energy source and that the PPi binding and/or catalytic site evolved to the present form of enzymes that utilize ATP. Finally, the detailed understanding of the PPi- utilizing enzymes that are crucial to Giardia but absent in the host, may be the targets and basis for the rational design of chemotherapeutic drugs against Giardial infections.